The study of DNA sequence variation is essential for many areas of research. The study of germ-line variations is essential for assessing the role of inheritance in normal and abnormal physiologic states (1). Other variations, developed somatically, are responsible for neoplasia (2). The identification of such mutations in urine, sputum, and stool can therefore be used for the detection of presymptomatic cancers (3-5). Similarly, the detection of somatic mutations in lymph nodes, blood, or bone marrow can provide data about the stage of disease, prognosis, and appropriateness of various therapies (5). Somatic mutations in non-neoplastic cells also occur and appear to accumulate as humans age or are exposed to environmental hazards (6). Such mutations occur in only a small fraction of the cells in a tissue, thereby complicating their analysis.
Central to the investigation of many of these issues is the detection and quantification of sequence variants within a population of DNA molecules. The number of molecules in each such collection is finite and therefore countable. Consider, for example, a collection of red and green balls. Counting these balls is simple in principle but subject to basic probability theory. If there is only one red ball for every 500 green balls, then it is necessary to count several thousand balls to get an accurate estimate of the proportion of red balls. If it is difficult to count enough balls to make a reliable estimate, one can elute the paint off all the balls and measure the color of the resultant paint mix.
In analogous fashion, small numbers of DNA molecules that vary by subtle changes (single base pair substitutions or small deletions or insertions) can be directly counted by amplifying individual DNA molecules (single molecule PCR) (7-12). Such digital techniques have been shown to be extremely useful for measuring variation in genes or their transcripts. But digital technologies have so far been limited to counting tens to thousands of molecules, either in the wells of microtiter plates, on microscope slides, or after electrophoresis of individual PCR products. Analog techniques, analogous to the elution of paint from the balls described above, are generally easier to implement and can assess millions of molecules simultaneously (13). However, their accuracy and sensitivity is limited by instrumental and experimental noise. There is a continuing need in the art for methods which are accurate and sensitive for measuring variation in genes or their transcripts.